Since their initial discovery, the number of different CRISPR-Cas systems has expanded rapidly 13,20,21 . Currently, CRISPR-Cas systems can be divided, according to evolutionary relationships, into two classes, six types and several subtypes 20 . The classes of CRISPR-Cas system are defined by the nature of the ribonucleoprotein effector complex: class 1 systems are characterized by a complex of multiple effector proteins, and class 2 systems encompass a single

Direct reprogramming by forced expression of transcription factors can convert one cell type into another. Thus, desired cell types can be generated bypassing pluripotency. However, direct reprogramming towards renal cells remains an unmet challenge. Here, we identify renal cell fate-inducing factors on the basis of their tissue specificity and evolutionarily conserved expression, and demonstrate that combined expression of Emx2, Hnf1b, Hnf4a and Pax8 converts mouse and human fibroblasts into induced renal tubular epithelial cells (iRECs). iRECs exhibit epithelial features, a global gene expression profile resembling their native counterparts, functional properties of differentiated renal tubule cells and sensitivity to nephrotoxic substances. Furthermore, iRECs integrate into kidney organoids and form tubules in decellularized kidneys. Our approach demonstrates that reprogramming factors can be identified by targeted in silico analysis. Renal tubular epithelial cells generated ex vivo by forced expression of transcription factors may facilitate disease modelling, drug and nephrotoxicity testing, and regenerative approaches.

Oseltamivir is routinely used worldwide for the treatment of severe influenza A virus infection, and should drug-resistant pandemic 2009 H1N1 viruses become widespread, this potent defense strategy might fail. Oseltamivir-resistant variants of the pandemic 2009 H1N1 influenza A virus have been detected in a substantial number of patients, but to date, the mutant viruses have not moved into circulation in the general population. It is not known whether the resistance mutations in viral neuraminidase (NA) reduce viral fitness. We addressed this question by studying transmission of oseltamivir-resistant mutants derived from two different isolates of the pandemic H1N1 virus in both the guinea pig and ferret transmission models. In vitro, the virus readily acquired a single histidine-to-tyrosine mutation at position 275 (H275Y) in viral neuraminidase when serially passaged in cell culture with increasing concentrations of oseltamivir. This mutation conferred a high degree of resistance to oseltamivir but not zanamivir. Unexpectedly, in guinea pigs and ferrets, the fitness of viruses with the H275Y point mutation was not detectably impaired, and both wild-type and mutant viruses were transmitted equally well from animals that were initially inoculated with 1:1 virus mixtures to naïve contacts. In contrast, a reassortant virus containing an oseltamivir-resistant seasonal NA in the pandemic H1N1 background showed decreased transmission efficiency and fitness in the guinea pig model. Our data suggest that the currently circulating pandemic 2009 H1N1 virus has a high potential to acquire drug resistance without losing fitness.

In organ transplantation, infection and rejection are major causes of graft loss linked by the net state of immunosuppression. To diagnose and treat these conditions earlier, and to improve long-term patient outcomes, refined strategies for the monitoring of patients after graft transplantation are needed. Here, we show that a fast and inexpensive assay based on CRISPR-Cas13 accurately detects BK polyomavirus DNA and cytomegalovirus DNA from patient-derived blood and urine samples, as well as CXCL9 mRNA (a marker of graft rejection) at elevated levels in urine samples from patients experiencing acute renal-transplant rejection. The assay, which we adapted for lateral-flow readout, enables via simple visualization the post-transplantation monitoring of common opportunistic viral infections and of graft rejection, and should facilitate point-of-care posttransplantation monitoring.